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| United States Patent |
5,773,292
|
|
Bander
|
June 30, 1998
|
Antibodies binding portions, and probes recognizing an antigen of
prostate epithelial cells but not antigens circulating in the blood
Abstract
The present invention is directed to the use of antibodies or binding
portions thereof or probes which recognize an antigen of normal, benign,
hyperplastic, and cancerous prostate epithelial cells or portions thereof.
These antibodies or binding portions thereof or probes can be labeled and
used for detection of such cells. They also can be used alone or bound to
a substance effective to ablate or kill such cells as a therapy for
prostate cancer. Also disclosed is a hybridoma cell line which produces a
monoclonal antibody recognizing antigens of normal, benign, hyperplastic,
and cancerous prostate epithelial cells or portions thereof.
| Inventors:
|
Bander; Neil H. (Chappaqua, NY)
|
| Assignee:
|
Cornell University (Ithaca, NY)
|
| Appl. No.:
|
463500 |
| Filed:
|
June 5, 1995 |
| Current U.S. Class: |
435/332; 424/138.1; 424/141.1; 424/152.1; 424/155.1; 435/7.21; 435/7.23; 530/387.5; 530/387.7; 530/388.2; 530/388.8; 530/389.1; 530/389.7; 530/391.1; 530/391.3; 530/391.7 |
| Intern'l Class: |
C07K 016/28; A61K 039/395; C12N 005/12; G01N 033/53 |
| Field of Search: |
530/387.1,387.7,388.1,388.8,389.1,389.7,391.3,391.7
436/501,503,504
435/7.1-7.95,70.21,172.2,240.27,333,7.21,7.23
424/138.1,141.1,155.1
|
References Cited
U.S. Patent Documents
| Re33405 | Oct., 1990 | Chu et al.
| |
| 4446122 | May., 1984 | Chu et al.
| |
| 4675287 | Jun., 1987 | Reisfeld et al.
| |
| 4840915 | Jun., 1989 | Bogoch.
| |
| 4863851 | Sep., 1989 | McEwan et al.
| |
| 4902615 | Feb., 1990 | Freeman et al.
| |
| 4970299 | Nov., 1990 | Bazinet et al.
| |
| 5055404 | Oct., 1991 | Ueda et al.
| |
| 5118611 | Jun., 1992 | Smith et al.
| |
| 5130129 | Jul., 1992 | Pardridge.
| |
| 5135737 | Aug., 1992 | Keana.
| |
| 5242824 | Sep., 1993 | Hellstrom et al.
| |
| 5250297 | Oct., 1993 | Grauer et al.
| |
| 5367060 | Nov., 1994 | Vandlen et al.
| |
Other References
Webb, Cancer Immunol Immunother 17:7-17, 1984.
Raynor JNCI 73:617-623, 1984.
Raynor The Prostate 9:21-31, 1986.
Ware Cancer Research 42:1215-1222, 1982.
Carroll Clin. Immunol Immunopathol 33:268-281, 1984.
Lopes Cancer Research 50:6423-6429 1990.
Starling Cancer Research 46:367-374 1986.
Theyer J. Urology 150: 1544-1547, 1993.
Leroy, Cancer 64: 1-5 1989.
Seaver, Gen. Eng. News V14, pp. 10-21, Aug. 1994.
Colher, Methods in Enzymology 121: 802-816 1986.
Bayer, Clinical Laboratory Methods, 9th ed., St. Louis: C. V. Mosby
Company, Chapter 35, "Clinical Serology," pp. 1025-1063 (1982).
Raynor et al., "Localization of a Normal Prostatic Secretory Product Using
the Monoclonal Antibody, KR-P8," Federation Proceedings, 44:793 (1985).
Raynor et al., "Localization of a Normal Prostatic Secretory Product Using
the Monoclonal Antibody, KR-P8," Journal of Urology, 134:384-387 (1985).
Guinan et al., "An Evaluation of Prostate Specific Antigen in Prostatic
Cancer," Journal of Urology, 137:686-689 (1987).
Guinan et al., "Methods of Early Diagnosis in Genitourinary Cancer,"
Cancer, 60:668-676 (1987).
Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor, New
York:Cold Spring Harbor Laboratory, Chapter 14, "Immunoassays," pp.
553-612 (1988).
Vitetta et al., "Redesigning Nature's Poisons top Create Anti-Tumor
Reagents," Science, 238:1098-1104 (1987).
Dillman, "Monoclonal Antibodies for Treating Cancer," Annals of Internal
Medicine, 111:592-602 (1989).
Waldmann, "Monoclonal Antibodies in Diagnosis and Therapy," Science,
252:1657-1662 (1991).
|
Primary Examiner: Feisee; Lila
Assistant Examiner: Johnson; Nancy A.
Attorney, Agent or Firm: Nixon, Hargrave, Devans & Doyle
Claims
What is claimed:
1. An isolated antibody or binding portion thereof recognizing an antigen
of normal, benign, hyperplastic, and cancerous prostate epithelial cells
or portions thereof but no antigens circulating in blood, said antibody or
binding portion thereof being immunoreactive with the prostate epithelial
cells at a level 200-500 fold greater than for other tissue, based on
immunohistochemical endpoint titrations, wherein said antibody or binding
portion thereof binds to the same epitope of the prostate-related antigen
as those to which monoclonal antibodies produced from hybridoma cell lines
having ATCC Designations HB 11424, HB 11425, HB 11426, HB 11427, HB 11892,
or HB 11893 bind.
2. An isolated antibody or binding portion thereof according to claim 1,
wherein the isolated antibody or binding portion thereof is an antibody
selected from the group consisting of a monoclonal antibody and a
polyclonal antibody.
3. An isolated antibody or binding portion thereof according to claim 2,
wherein the antibody is a monoclonal antibody produced from a hybridoma
cell line selected from the group consisting of the hybridoma cell lines
having ATCC Designations HB 11424, HB 11425, HB 11426, HB 11427, HB 11892,
and HB 11893.
4. An isolated antibody or binding portion thereof according to claim 2,
wherein the isolated antibody or binding portion thereof is a binding
portion of an antibody selected from the group consisting of a Fab
fragment, a F(ab').sub.2 fragment, and a Fv fragment.
5. An isolated antibody or binding portion thereof according to claim 1,
wherein the antibody or binding portion thereof is bound to a cytotoxic
drug.
6. An isolated antibody or binding portion thereof according to claim 5,
wherein the cytotoxic drug is selected from the group consisting of a
therapeutic drug, a compound emitting radiation, molecules of plant,
fungal, or bacterial origin, biological proteins, and mixtures thereof.
7. A composition comprising:
an antibody or binding portion thereof according to claim 5 and
a physiological acceptable carrier, excipient, or stabilizer mixed with the
antibody or binding portion thereof.
8. A composition comprising:
an antibody or binding portion thereof according to claim 5 and
a pharmaceutically acceptable carrier, excipient, or stabilizer mixed with
the antibody or binding portion thereof.
9. An isolated antibody or binding portion thereof according to claim 1,
wherein said antibody or binding portion thereof is bound to a detectable
label.
10. An isolated antibody or binding portion thereof according to claim 9,
wherein the label is selected from the group consisting of a fluorescent
label, a biologically-active enzyme label, a radioactive label, a nuclear
magnetic resonance active label, a luminescent label, and a chromophore
label.
11. A composition comprising:
an antibody or binding portion thereof according to claim 9 and
a physiological acceptable carrier, excipient, or stabilizer mixed with the
antibody or binding portion thereof.
12. A composition comprising:
an antibody or binding portion thereof according to claim 9 and
a pharmaceutically acceptable carrier, excipient, or stabilizer mixed with
the antibody or binding portion thereof.
13. A kit for detecting prostate cancer comprising:
an antibody or binding portion thereof according to claim 9 and
means to detect the label.
14. A kit according to claim 13, wherein the label is selected from the
group consisting of a fluorescent label, a biologically-active enzyme
label, a radioactive label, a nuclear magnetic resonance active label, a
luminescent label, and a chromophore label.
15. A kit according to claim 13, wherein said antibody or binding portion
thereof is a monoclonal antibody produced from a hybridoma cell line
selected from the group consisting of the hybridoma cell lines having ATCC
Designations HB 11424, HB 11425, HB 11426, HB 11427, HB 11892, and HB
11893.
16. A kit according to claim 13, wherein the antibody or binding portion
thereof is in a composition further comprising a physiological acceptable
carrier, excipient, or stabilizer.
17. A kit according to claim 13, wherein the antibody or binding portion
thereof is in a composition further comprising a pharmaceutically
acceptable carrier, excipient, or stabilizer.
18. A hybridoma cell line that produces a monoclonal antibody recognizing
an antigen of normal, benign, hyperplastic, and cancerous prostate
epithelial cells or portions thereof but no antigens circulating in blood,
wherein said monoclonal antibody binds to the same epitope of the
prostate-related antigen as those to which monoclonal antibodies produced
from hybridoma cell lines having ATCC Designations HB 11424, HB 11425, HB
11426, HB 11427, HB 11892, or HB 11893 bind.
19. An isolated antibody or binding portion thereof according to claim 1
which recognizes an antigen found in all normal, benign, hyperplastic, and
cancerous prostate epithelial cells or portions thereof.
Description
FIELD OF THE INVENTION
The present invention relates to the treatment and diagnosis of prostate
cancer with antibodies or binding portions thereof.
BACKGROUND OF THE INVENTION
Prostate cancer is the most common cancer in men with an estimated 244,000
cases in 1995 in the United States. It is the second leading cause of
death among men who die from neoplasia with an estimated 44,000 deaths per
year. Prompt detection and treatment is needed to limit mortality caused
by prostate cancer.
Detection of Prostate Cancer
When it metastasizes, prostatic cancer has a distinct predilection for bone
and lymph nodes. Saitoh, H., et al., "Metastatic Patterns of Prostatic
Cancer. Correlation Between Sites And Number Of Organs Involved." Cancer,
54:3078-3084 (1984). At the time of clinical diagnosis, as many as 25% of
patients have bone metastasis demonstrable by radionuclide scans. Murphy,
G. P., et al., "The National Survey Of Prostate Cancer In The United
States By The American College Of Surgeons," J. Urol., 127:928-939 (1982).
Accurate clinical evaluation of nodal involvement has proven to be
difficult. Imaging techniques such as computed tomography ("CT") or
magnetic resonance ("MR") imaging are unable to distinguish metastatic
prostate cancer involvement of lymph nodes by criterion other than size
(i.e., >1 cm). Therefore, by definition, these imaging modalities are
inherently insensitive in the detection of small volume (<1 cm) disease as
well as non-specific in the detection of larger volume adenopathy. A
recent study assessed the accuracy of MR in patients with clinically
localized prostate cancer. Rifkin, M. D., et al., "Comparison Of Magnetic
Resonance Imaging And Ultrasonography In Staging Early Prostate Cancer,"
N. Engl. J. Med., 323:621-626 (1990). In this study, 194 patients
underwent an MR and 185 of these patients had a lymph node dissection. 23
(13w) patients had pathologically involved lymph nodes. MR was suspicious
in only 1 of these 23 cases resulting in a sensitivity of 4%. Similar
results have also been noted with CT scans. Gasser, T. C., et al., "MRI
And Ultrasonography In Staging Prostate Cancer," N. Engl. J. Med.
(Correspondence), 324(7):49-495 (1991).
The elevation of serum acid phosphatase activity in patients having
metastasized prostate carcinoma was first reported by Gutman et al., J.
Clin. Invest 17:473 (1938). In cancer of the prostate, prostatic acid
phosphatase is released from the cancer tissue into the blood stream with
the result that the total serum acid phosphatase level can be greatly
increased above normal values. Numerous studies of this enzyme and its
relation to prostatic cancer have been made since that time, e.g. Yam,
Amer. J. Med. 56:604 (1974). However, the measurement of serum acid
phosphatase is elevated in about 65-90 percent of patients having
carcinoma of the prostate with bone metastasis; in about 30 percent of
patients without roentgenological evidence of bone metastasis; and in
about only 5-10 percent of patients lacking clinically demonstrable
metastasis.
Prior art attempts to develop a specific test for prostatic acid
phosphatase have met with only limited success, because techniques which
rely on enzyme activity on a so-called "specific" substrate cannot take
into account other biochemical and immunochemical differences among the
many acid phosphatases which are unrelated to enzyme activity of prostate
origin. In the case of isoenzymes, i.e. genetically defined enzymes having
the same characteristic enzyme activity and a similar molecular structure
but differing in amino acid sequences and/or content and, therefore,
immunochemically distinguishable, it would appear inherently impossible to
distinguish different isoenzyme forms merely by the choice of a particular
substrate. It is, therefore, not surprising that none of these prior art
methods is highly specific for the direct determination of prostatic acid
phosphatase activity; e.g. see Cancer 5:236 (1952); J. Lab. Clin. Med.
82:486 (1973); Clin. Chem. Acta. 44:21 (1973); and J. Physiol. Chem.
356:1775 (1975).
In addition to the aforementioned problems of non-specificity which appear
to be inherent in many of the prior art reagents employed for the
detection of prostate acid phosphatase, there have been reports of
elevated serum acid phosphatase associated with other diseases, which
further complicates the problem of obtaining an accurate clinical
diagnosis of prostatic cancer. For example, Tuchman et al., Am. J. Med.
27:959 (1959) noted that serum acid phosphatase levels appear to be
elevated in patients with Gaucher's disease.
Due to the inherent difficulties in developing a "specific" substrate for
prostrate acid phosphatase, several researchers have developed
immunochemical methods for the detection of prostate acid phosphatase.
However, the previously reported immunochemical methods have drawbacks of
their own which have precluded their widespread acceptance. For example,
Shulman et al., Immunology 93:474 (1964) described an immuno-diffusion
test for the detection of human prostate acid phosphatase. Using antisera
prepared from a prostatic fluid antigen obtained by rectal massage from
patients with prostatic disease, no cross-reactivity precipitin line was
observed in the double diffusion technique against extracts of normal
kidney, testicle, liver, and lung. However, this method has the
disadvantages of limited sensitivity, even with the large amounts of
antigen employed, and of employing antisera which may cross-react with
other, antigenically unrelated serum protein components present in
prostatic fluid.
WO 79/00475 to Chu et. al. describes a new method for the detection of
prostatic acid phosphatase isoenzyme patterns associated with prostatic
cancer which obviates many of the above drawbacks. However, practical
problems are posed by the need for a source of cancerous prostate tissue
from which the diagnostically relevant prostatic acid phosphatase
isoenzyme patterns associated with prostatic cancer are extracted for the
preparation of antibodies thereto.
In recent years, considerable effort has been spent to identify enzyme or
antigen markers for various types of malignancies with the view towards
developing specific diagnostic reagents. The ideal tumor marker would
exhibit, among other characteristics, tissue or cell-type specificity, and
would be released into the circulation or other biological milieu which is
easily obtained from individuals. Previous investigators have demonstrated
the occurrence of human prostrate tissue-specific antigens.
Treatment of Prostate Cancer
As described in W. J. Catalona, "Management of Cancer of the Prostate," New
Engl. J. Med. 331(15):996-1004 (1994), the management of prostate cancer
can be achieved by watchful waiting, curative treatment, and palliation.
For men with a life expectancy of less than 10 years, watchful waiting is
appropriate where low-grade, low-stage prostate cancer is discovered at
the time of a partial prostatectomy for benign hyperplasia. Such cancers
rarely progress during the first five years after detection. On the other
hand, for younger men, curative treatment is often more appropriate.
Where prostate cancer is localized and the patient's life expectancy is 10
years or more, radical prostatectomy offers the best chance for
eradication of the disease. Historically, the drawback of this procedure
is that most cancers had spread beyond the bounds of the operation by the
time they were detected. However, the use of prostate-specific antigen
testing has permitted early detection of prostate cancer. As a result,
surgery is less expensive with fewer complications. Patients with bulky,
high-grade tumors are less likely to be successfully treated by radical
prostatectomy.
After surgery, if there are detectable serum prostate-specific antigen
concentrations, persistent cancer is indicated. In many cases,
prostate-specific antigen concentrations can be reduced by radiation
treatment. However, this concentration often increases again within two
years.
Radiation therapy has also been widely used as an alternative to radical
prostatectomy. Patients generally treated by radiation therapy are those
who are older and less healthy and those with higher-grade, more
clinically advanced tumors. Particularly preferred procedures are
external-beam therapy which involves three dimensional, conformal
radiation therapy where the field of radiation is designed to conform to
the volume of tissue treated, and interstitial-radiation therapy where
seeds of radioactive compounds are implanted using ultrasound guidance.
For treatment of patients with locally advanced disease, hormonal therapy
before or following radical prostatectomy or radiation therapy has been
utilized. Hormonal therapy is the main form of treating men with
disseminated prostate cancer. Orchiectomy reduces serum testosterone
concentrations, while estrogen treatment is similarly beneficial.
Diethylstilbestrol from estrogen is another useful hormonal therapy which
has a disadvantage of causing cardiovascular toxicity. When
gonadotropin-releasing hormone agonists are administered testosterone
concentrations are ultimately reduced. Flutamide is a nonsteroidal,
anti-androgen agent that blocks binding of testosterone to its
intracellular receptors. As a result, it blocks the effect of
testosterone, increasing serum testosterone concentrations and allows
patients to remain potent--a significant problem after radical
prostatectomy and radiation treatments.
Cytotoxic chemotherapy is largely ineffective in treating prostate cancer.
Its toxicity makes such therapy unsuitable for elderly patients. In
addition, prostate cancer is relatively resistant to cytotoxic agents.
Use of Monoclonal Antibodies in Prostate Cancer Detection and Treatment
Theoretically, radiolabeled monoclonal antibodies ("mAbs") offer the
potential to enhance both the sensitivity and specificity of detecting
prostatic cancer within lymph nodes and elsewhere. While many mAbs have
previously been prepared against prostate related antigens, none of these
mAbs were specifically generated with an imaging objective in mind.
Nevertheless, the clinical need has led to evaluation of some of these
mAbs as possible imaging agents. Vihko, P., et al., "Radioimaging of
Prostatic Carcinoma With Prostatic Acid Phosphatase--Specific Antibodies,"
Biotechnology in Diagnostics, 131-134 (1985); Babaian, R. J., et al.,
"Radioimmunological Imaging of Metastatic Prostatic Cancer With
111-Indium-Labeled Monoclonal Antibody PAY 276," J. Urol., 137:439-443
(1987); Leroy, J M., et al., "Radioimmunodetection Of Lymph Node Invasion
In Prostatic Cancer. The Use Of Iodine 123 (123-I)-Labeled Monoclonal
Anti-Prostatic Acid Phosphatase (PAP) 227 A F (ab') 2 Antibody Fragments
In Vivo," Cancer, 64:1-5 (1989); Meyers, J. F., et al., "Development Of
Monoclonal Antibody Imaging Of Metastatic Prostatic Carcinoma," The
Prostate, 14:209-220 (1989).
In some cases, the monoclonal antibodies developed for detection and/or
treatment of prostate cancer recognize antigens specific to malignant
prostatic tissues. Such antibodies are thus used to distinguish malignant
prostatic tissue (for treatment or detection) from benign prostatic
tissue. See U.S. Pat. No. 4,970,299 to Bazinet et al. and U.S. Pat. No.
4,902,615 to Freeman et al.
Other monoclonal antibodies react with surface antigens on all prostate
epithelial cells whether cancerous or benign. See U.S. Pat. No. 4,446,122
and Re 33,405 to Chu et al., U.S. Pat. No. 4,863,851 to McEwan et al., and
U.S. Pat. No. 5,055,404 to Ueda et al. However, the antigens detected by
these monoclonal antibodies are present in the blood and, therefore,
compete with antigens at tumor sites for the monoclonal antibodies. This
causes background noise which makes the use of such antibodies
inappropriate for in vivo imaging. In therapy, such antibodies, if bound
to a cytotoxic agent, could be harmful to other organs.
The present invention is directed to overcoming the deficiencies of prior
art antibodies in diagnosing and treating prostate cancer.
SUMMARY OF THE INVENTION
One aspect of the present invention relates to a method of ablating or
killing normal, benign, hyperplastic, and cancerous prostate epithelial
cells. The process involves providing an antibody or binding portion
thereof or probe which recognizes an antigen (such as a surface antigen)
of such cells but substantially no antigens circulating in the blood. The
antibody or binding portion thereof or probe can be used alone or is bound
to a substance effective to kill the cells upon binding of the antibody or
binding portion thereof or probe to the cells. These antibodies or binding
portions thereof or probes are then contacted with the cells under
conditions effective to permit both binding of the antibody or binding
portion thereof or probe to the antigens and killing or ablating of the
cells.
Another aspect of the present invention relates to a method of detecting
normal, benign, hyperplastic, and cancerous epithelial cells or portions
thereof in a biological sample. This method involves providing an antibody
or binding portion thereof or probe which recognizes an antigen of the
cells but substantially no antigens circulating in the blood. The antibody
or binding portion thereof or probe is bound to a label effective to
permit detection of the cells or portions thereof upon binding of the
antibody or binding portion thereof or probe to the cells or portions
thereof. The biological sample is contacted with the antibody or binding
portion thereof or probe having a label under conditions effective to
permit binding of the antibody or binding portion thereof or probe to the
antigen of any of the cells or portions thereof in the biological sample.
The presence of any cells or portions thereof in the biological sample is
detected by detection of the label.
Another aspect of the present invention pertains to an isolated antibody or
binding portion thereof or probe recognizing an antigen of normal, benign,
hyperplastic, and cancerous prostate epithelial cells or portions thereof
but substantially no antigens circulating in the blood. A hybridoma cell
line that produces monoclonal antibodies of this type and an antigen
recognized by these monoclonal antibodies are also disclosed.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A, 1B, 1C and 1D show immunohistochemical staining of benign
prostate hyperplasia (FIGS. 1A and C) and prostate cancer (FIGS. 1B and D)
with Prost 30 (FIGS. 1A and B) and Prost 410 (FIGS. 1C and D). Epithelial
cells and luminal spaces were strongly stained. Prost 30 demonstrates
enhanced immunoreactivity at the cell surface. Magnification.times.350
(FIGS. 1A, C and D), .times.175 (FIG. 1B).
FIGS. 2A and 2B show immunohistochemical staining of BPH (i.e. prostatic
epithelium) sections by mAb Prost 130 (FIG. 2A) and Prost 185 (FIG. 2B) at
5 ug/ml. Magnification.times.350.
FIGS. 3A and 3B shows a sandwich ELISA in which Prost 130 (FIG. 3A) or
Prost 185 (FIG. 3B) were coated on Terasaki plates with coating buffer
overnight at 37.degree. C. After adding solubilized prostate antigens,
biotin conjugated mAbs were added. Prost 130-biotin and Prost 185-biotin
reacted with antigens captured by Prost 130 (FIG. 3A). Prost 130 was
inhibited by Prost 130 (FIG. 3A). Prost 130-biotin reacted with antigens
captured by Prost 185, but Prost 185-biotin did not.
FIGS. 4A and 4B show an inhibition assay. In FIG. 4A, binding of Prost
130-biotin to antigens captured by Prost 130 was inhibited by Prost 130
but not by Prost 185. In FIG. 4B, binding of Prost 185-biotin to antigens
captured by Prost 130 was inhibited by Prost 185 but not by Prost 130.
FIG. 5 shows a resected prostate with two adjacent tubes of blood at right
angles to each other. The latter was drawn at the same time as the
prostate was resected--i.e., one week after 131I-Prost 30 administration.
The color intensity is directly proportional to the radioactivity. This
figure shows that the radiolabeled antibody 1) localizes to the prostate
and 2) actually concentrates in the prostate at far higher levels than the
blood and remains in the prostate for >one week.
DETAILED DESCRIPTION OF THE INVENTION
One aspect of the present invention relates to a method of ablating or
killing normal, benign, hyperplastic, and cancerous prostate epithelial
cells. The process involves providing an antibody or binding portion
thereof or probe which recognizes an antigen (such as a surface antigen)
of such cells but substantially no antigens circulating in the blood. The
antibody or binding portion thereof or probe can be used alone or is bound
to a substance effective to kill the cells upon binding of the antibody or
binding portion thereof or probe to the cells. These antibodies or binding
portions thereof or probes are then contacted with the cells under
conditions effective to permit both binding of the antibody or binding
portion thereof or probe to the antigens and killing or ablating of the
cells. In its preferred form, such contacting is carried out in a living
mammal by administering the antibody or binding portion thereof or probe
to the mammal under conditions effective to permit both binding of the
antibody or binding portion thereof or probe to the antigens and killing
or ablating of the cells. Such administration can be carried out orally or
parenterally.
Another aspect of the present invention relates to a method of detecting
normal, benign, hyperplastic, and cancerous epithelial cells or portions
thereof in a biological sample. This method involves providing an antibody
or binding portion thereof or probe which recognizes an antigen of the
cells but substantially no antigens circulating in the blood. The antibody
or binding portion thereof or probe is bound to a label effective to
permit detection of the cells or portions thereof upon binding of the
antibody or binding portion thereof or probe to the cells or portions
thereof. The biological sample is contacted with the antibody or binding
portion thereof or probe having a label under conditions effective to
permit binding of the antibody or binding portion thereof or probe to the
antigen of any of the cells or portions thereof in the biological sample.
The presence of any cells or portions thereof in the biological sample is
detected by detection of the label. In its preferred form, such contacting
is carried out in a living mammal and involves administering the antibody
or binding portion thereof or probe to the mammal under conditions
effective to permit binding of the antibody or binding portion thereof or
probe to the antigen of any of the cells or portions thereof in the
biological sample. Again, such administration can be carried out orally or
parenterally. Alternatively, the contacting step can be carried out in a
sample of serum or urine or other body fluids.
Antibodies suitable for either killing, ablating, or detecting normal,
benign, hyperplastic, and cancerous prostate epithelial cells can be
monoclonal or polyclonal. In addition, antibody fragments,
half-antibodies, hybrid derivatives, and probes may be utilized. These
antibodies, binding portions thereof, or probes recognize cell antigens or
portions thereof in normal, benign, hyperplastic, and cancerous prostate
epithelial cells. However, these antibodies, binding portions thereof, or
probes bind to substantially no antigens in the blood. As a result,
binding of the antibodies or binding portions thereof or probes is
concentrated in areas where there are large numbers of prostate epithelial
cells or portions thereof.
Monoclonal antibody production may be effected by techniques which are
well-known in the art. Basically, the process involves first obtaining
immune cells (lymphocytes) from the spleen of a mammal (e.g., mouse) which
has been previously immunized with the antigen of interest either in vivo
or in vitro. The antibody-secreting lymphocytes are then fused with
(mouse) myeloma cells or transformed cells, which are capable of
replicating indefinitely in cell culture, thereby producing an immortal,
immunoglobulin-secreting cell line. The resulting fused cells, or
hybridomas, are cultured, and the resulting colonies screened for the
production of the desired monoclonal antibodies. Colonies producing such
antibodies are cloned, and grown either in vivo or in vitro to produce
large quantities of antibody. A description of the theoretical basis and
practical methodology of fusing such cells is set forth in Kohler and
Milstein, Nature 256:495 (1975), which is hereby incorporated by
reference.
Mammalian lymphocytes are immunized by in vivo immunization of the animal
(e.g., a mouse) with the protein or polypeptide of the present invention.
Such immunizations are repeated as necessary at intervals of up to several
weeks to obtain a sufficient titer of antibodies. Following the last
antigen boost, the animals are sacrificed and spleen cells removed.
Fusion with mammalian myeloma cells or other fusion partners capable of
replicating indefinitely in cell culture is effected by standard and
well-known techniques, for example, by using polyethylene glycol (PEG) or
other fusing agents (See Milstein and Kohler, Eur. J. Immunol. 6:511
(1976), which is hereby incorporated by reference). This immortal cell
line, which is preferably murine, but may also be derived from cells of
other mammalian species, including but not limited to rats and humans, is
selected to be deficient in enzymes necessary for the utilization of
certain nutrients, to be capable of rapid growth and to have good fusion
capability. Many such cell lines are known to those skilled in the art,
and others are regularly described.
Procedures for raising polyclonal antibodies are also well known.
Typically, such antibodies can be raised by administering the protein or
polypeptide of the present invention subcutaneously to New Zealand white
rabbits which have first been bled to obtain pre-immune serum. The
antigens can be injected at a total volume of 100 .mu.l per site at six
different sites. Each injected material will contain synthetic surfactant
adjuvant pluronic polyols, or pulverized acrylamide gel containing the
protein or polypeptide after SDS-polyacrylamide gel electrophoresis. The
rabbits are then bled two weeks after the first injection and periodically
boosted with the same antigen three times every six weeks. A sample of
serum is then collected 10 days after each boost. Polyclonal antibodies
are then recovered from the serum by affinity chromatography using the
corresponding antigen to capture the antibody. Ultimately, the rabbits are
euthenized with pentobarbital 150 mg/Kg IV. This and other procedures for
raising polyclonal antibodies are disclosed in E. Harlow, et. al.,
editors, Antibodies: A Laboratory Manual (1988), which is hereby
incorporated by reference.
In addition to utilizing whole antibodies, the processes of the present
invention encompass use of binding portions of such antibodies. Such
binding portions include Fab fragments, F(ab').sub.2 fragments, and Fv
fragments. These antibody fragments can be made by conventional
procedures, such as proteolytic fragmentation procedures, as described in
J. Goding, Monoclonal Antibodies: Principles and Practice, pp. 98-118
(N.Y. Academic Press 1983), which is hereby incorporated by reference.
Alternatively, the processes of the present invention can utilize probes
found either in nature or prepared synthetically by recombinant DNA
procedures or other biological procedures. Suitable probes are molecules
which bind to prostate-related antigens identified by the monoclonal
antibodies of the present invention. Such probes can be e.g., proteins,
peptides, lectins, or nucleic acid probes.
Here, it is preferred to utilize the monoclonal antibodies identified below
in Table 1:
TABLE 1
______________________________________
ATCC Designation for
Monoclonal Antibody Name
Hybridoma Cell Line
______________________________________
Prost 30 HB 11424
Prost 185 HB 11425
Prost 410 HB 11426
Prost 130 HB 11427
C37 HB 11892
C219 HB 11893
______________________________________
It is particularly desirable to utilize a mixture of these antibodies or
other antibodies to treat or image prostate epithelial cells with varying
surface antigen characteristics.
The present invention also relates to antigens of normal, benign,
hyperplastic, and cancerous prostate epithelial cells recognized by the
monoclonal antibodies in Table 1.
Regardless of whether the antibodies or binding portions thereof or probes
are used for treatment or therapy, they can be administered orally,
parenterally, subcutaneously, intravenously, intramuscularly,
intraperitoneally, by intranasal instillation, by intracavitory or
intravesical instillation, intraocularly, intraarterially,
intralesionally, or by application to mucous membranes, such as, that of
the nose, throat, and bronchial tubes. They may be administered alone or
with pharmaceutically or physiologically acceptable carriers, excipients,
or stabilizers, and can be in solid or liquid form such as, tablets,
capsules, powders, solutions, suspensions, or emulsions.
The solid unit dosage forms can be of the conventional type. The solid form
can be a capsule, such as an ordinary gelatin type containing the antibody
or binding portion thereof of the present invention and a carrier, for
example, lubricants and inert fillers such as, lactose, sucrose, or
cornstarch. In another embodiment, these compounds are tableted with
conventional tablet bases such as lactose, sucrose, or cornstarch in
combination with binders like acacia, cornstarch, or gelatin,
disintegrating agents such as, cornstarch, potato starch, or alginic acid,
and a lubricant like stearic acid or magnesium stearate.
The antibody or binding portion thereof or probes of the present invention
may also be administered in injectable dosages by solution or suspension
of these materials in a physiologically acceptable diluent with a
pharmaceutical carrier. Such carriers include sterile liquids such as
water and oils, with or without the addition of a surfactant and other
pharmaceutically and physiologically acceptable carrier, including
adjuvants, excipients or stabilizers. Illustrative oils are those of
petroleum, animal, vegetable, or synthetic origin, for example, peanut
oil, soybean oil, or mineral oil. In general, water, saline, aqueous
dextrose and related sugar solution, and glycols such as, propylene glycol
or polyethylene glycol, are preferred liquid carriers, particularly for
injectable solutions.
For use as aerosols, the antibody or binding portion thereof or probe of
the present invention in solution or suspension may be packaged in a
pressurized aerosol container together with suitable propellants, for
example, hydrocarbon propellants like propane, butane, or isobutane with
conventional adjuvants. The materials of the present invention also may be
administered in a non-pressurized form such as in a nebulizer or atomizer.
The antibodies or binding portions thereof or probes may be utilized to
detect normal, benign, hyperplastic, and cancerous prostate epithelial
cells in vivo. This is achieved by labeling the antibody or binding
portion thereof or probe administering the labeled antibody or binding
portion thereof or probe to a mammal, and then imaging the mammal.
Examples of labels useful for diagnostic imaging in accordance with the
present invention are radiolabels such as .sup.131 I, .sup.111 In,
.sup.123 I, .sup.99 mTc, .sup.32 P, .sup.125 I, .sup.3 H, .sup.14 C, and
.sup.188 Rh, fluorescent labels such as fluorescein and rhodamine, nuclear
magnetic resonance active labels, chemiluminescers such as luciferin, and
enzymatic markers such as peroxidase or phosphatase. The antibody or
binding portion thereof or probe can be labeled with such reagents using
techniques known in the art. For example, see Wensel and Meares,
Radioimmunoimaging and Radioimmunotherapy, Elsevier, N.Y. (1983), which is
hereby incorporated by reference, for techniques relating to the
radiolabeling of antibodies. See also, D. Colcher et al., "Use of
Monoclonal Antibodies as Radiopharmaceuticals for the Localization of
Human Carcinoma Xenografts in Athymic Mice", Meth. Enzymol. 121: 802-816
(1986), which is hereby incorporated by reference.
A radiolabeled antibody or binding portion thereof or probe of this
invention can be used for in vitro diagnostic tests. The specific activity
of a tagged antibody, binding portion thereof, or probe depends upon the
half-life, the isotopic purity of the radioactive label, and how the label
is incorporated into the antibody or binding portion thereof or probe.
Table 2 lists several commonly-used isotopes, their specific activities
and half-lives. In immunoassay tests, the higher the specific activity, in
general, the better the sensitivity.
TABLE 2
______________________________________
Specific Activity of Pure
Isotope Isotope (Curies/mole)
Half-Life
______________________________________
.sup.14 C
6.25 .times. 10.sup.1
5720 years
.sup.3 H
2.01 .times. 10.sup.4
12.5 years
.sup.35 S
1.50 .times. 10.sup.6
87 days
.sup.125 I
2.18 .times. 10.sup.6
60 days
.sup.32 P
3.16 .times. 10.sup.6
14.3 days
.sup.131 I
1.62 .times. 10.sup.7
8.1 days
______________________________________
Procedures for labeling antibodies, binding portions thereof, or probes
with the radioactive isotopes listed in Table 2 are generally known in the
art. Tritium labeling procedures are described in U.S. Pat. No. 4,302,438,
which is hereby incorporated by reference. Iodinating, tritium labeling,
and .sup.35 S labeling procedures especially adapted for murine monoclonal
antibodies are described by Goding, J. W. (supra, pp 124-126) and the
references cited therein, which are hereby incorporated by reference.
Other procedures for iodinating antibodies, binding portions thereof, or
probes are described by Hunter and Greenwood, Nature 144:945 (1962), David
et al., Biochemistry 13:1014-1021 (1974), and U.S. Pat. Nos. 3,867,517 and
4,376,110, which are hereby incorporated by reference. Radiolabeling
elements which are useful in imaging include .sup.123 I, .sup.131 I,
.sup.111 In, and .sup.99m TC for example. Procedures for iodinating
antibodies, binding portions thereof, or probes are described by
Greenwood, F. et al., Biochem. J. 89:114-123 (1963); Marchalonis, J.,
Biochem. J. 113:299-305 (1969); and Morrison, M. et al., Immunochemistry,
289-297 (1971), which are hereby incorporated by reference. Procedures for
.sup.99m Tc-labeling are described by Rhodes, B. et al. in Burchiel, S. et
al. (eds.), Tumor Imaging: The Radioimmunochemical Detection of Cancer,
New York: Masson 111-123 (1982) and the references cited therein, which
are hereby incorporated by reference. Procedures suitable for .sup.111
In-labeling antibodies, binding portions thereof, or probes are described
by Hnatowich, D. J. et al., J. Immul. Methods, 65:147-157 (1983),
Hnatowich, D. et al., J. Applied Radiation, 35:554-557 (1984), and
Buckley, R. G. et al., F. E. B. S. 166:202-204 (1984), which are hereby
incorporated by reference.
In the case of a radiolabeled antibody, binding portion thereof, or probe,
the antibody, binding portion thereof, or probe is administered to the
patient, is localized to the tumor bearing the antigen with which the
antibody, binding portion thereof, or probe reacts, and is detected or
"imaged" in vivo using known techniques such as radionuclear scanning
using e.g., a gamma camera or emission tomography. See e.g., A. R.
Bradwell et al., "Developments in Antibody Imaging", Monoclonal Antibodies
for Cancer Detection and Therapy, R. W. Baldwin et al., (eds.), pp. 65-85
(Academic Press 1985), which is hereby incorporated by reference.
Alternatively, a positron emission transaxial tomography scanner such as
designated Pet VI located at Brookhaven National Laboratory can be used
where the radiolabel emits positrons (e.g., .sup.11 C, .sup.18 F, .sup.15
O and .sup.13 N).
Fluorophore and chromophore labeled antibodies, binding portions thereof,
or probes can be prepared from standard moieties known in the art. Since
antibodies and other proteins absorb light having wavelengths up to about
310 nm, the fluorescent moieties should be selected to have substantial
absorption at wavelengths above 310 nm and preferably above 400 nm. A
variety of suitable fluorescers and chromophores are described by Stryer,
Science, 162:526 (1968) and Brand, L. et al., Annual Review of
Biochemistry, 41:843-868 (1972), which are hereby incorporated by
reference. The antibodies, binding portions thereof, or probes can be
labeled with fluorescent chromophore groups by conventional procedures
such as those disclosed in U.S. Pat. Nos. 3,940,475, 4,289,747, and
4,376,110, which are hereby incorporated by reference.
One group of fluorescers having a number of the desirable properties
described above are the xanthene dyes, which include the fluoresceins
derived from 3,6-dihydroxy-9-henylxanthhydrol and resamines and rhodamines
derived from 3,6-diamino-9-phenylxanthydrol and lissanime rhodamine B. The
rhodamine and fluorescein derivatives of 9-o-carboxyphenylxanthhydrol have
a 9-o-carboxyphenyl group. Fluorescein compounds having reactive coupling
groups such as amino and isothiocyanate groups such as fluorescein
isothiocyanate and fluorescamine are readily available. Another group of
fluorescent compounds are the naphthylamines, having an amino group in the
.alpha. or .beta. position.
Antibodies or binding portions thereof or probes can be labeled with
fluorchromes or chromophores by the procedures described by Goding, J.
(supra, pp 208-249). The antibodies or binding portions thereof or probes
can be labeled with an indicating group containing the NMR-active .sup.19
F atom, or a plurality of such atoms inasmuch as (i) substantially all of
naturally abundant fluorine atoms are the .sup.19 F isotope and, thus,
substantially all fluorine-containing compounds are NMR-active; (ii) many
chemically active polyfluorinated compounds such as trifluoracetic
anhydride are commercially available at relatively low cost, and (iii)
many fluorinated compounds have been found medically acceptable for use in
humans such as the perfluorinated polyethers utilized to carry oxygen as
hemoglobin replacements. After permitting such time for incubation, a
whole body NMR determination is carried out using an apparatus such as one
of those described by Pykett, Scientific American, 246:78-88 (1982), which
is hereby incorporated by reference, to locate and image prostate
epithelial cells.
The antibodies or binding portions thereof or probes can also be utilized
to kill or ablate normal, benign, hyperplastic, and cancerous prostate
epithelial cells in vivo. This involves using the antibodies or binding
portions thereof or probes by themselves or with a cytotoxic drug, which
the antibodies, binding portions thereof, or probes to normal, benign,
hyperplastic, and cancerous prostate epithelial cells where those cells
are ablated or killed. This involves administering the antibodies or
binding portions thereof or probes bonded to a cytotoxic drug to a mammal
requiring such treatment. Since the antibodies or binding portions thereof
or probes recognize prostate epithelial cells, any such cells to which the
antibodies or binding portions thereof or probes bind are destroyed.
Although such administration may destroy normal prostate epithelial cells,
this is not problematic, because the prostate is not required for life or
survival. Although the prostate may indirectly contribute to fertility,
this is not likely to be a practical consideration in patients receiving
the treatment of the present invention.
The antibodies or binding portions thereof or probes of the present
invention may be used to deliver a variety of cytotoxic drugs including
therapeutic drugs, a compound emitting radiation, molecules of plants,
fungal, or bacterial origin, biological proteins, and mixtures thereof.
Enzymatically active toxins and fragments thereof are exemplified by
diphtheria toxin A fragment, nonbinding active fragments of diphtheria
toxin, exotoxin A (from Pseudomonas aeruginosa), ricin A chain, abrin A
chain, modeccin A chain, .alpha.-sacrin, certain Aleurites fordii
proteins, certain Dianthin proteins, Phytolacca americana proteins (PAP,
PAPII and PAP-S), Morodica charantia inhibitor, curcin, crotin, Saponaria
officinalis inhibitor, gelonin, mitogillin, restrictocin, phenomycin, and
enomycin, for example. Procedures for preparing enzymatically active
polypeptides of the immunotoxins are described in W084/03508 and
W085/03508, which are hereby incorporated by reference. Certain cytotoxic
moieties are derived from adriamycin, chlorambucil, daunomycin,
methotrexate, neocarzinostatin, and platinum, for example.
Procedures for conjugating the antibodies or binding portions thereof or
probes with the cytotoxic agents have been previously described.
Procedures for conjugating chlorambucil with antibodies are described by
Flechner, I,. European Journal of Cancer, 9:741-745 (1973); Ghose, T. et
al., British Medical Journal, 3:495-499 (1972); and Szekerke, M., et al.,
Neoplasma, 19:211-215 (1972), which are hereby incorporated by reference.
Procedures for conjugating daunomycin and adriamycin to antibodies are
described by Hurwitz, E. et al., Cancer Research, 35:1175-1181 (1975) and
Arnon, R. et al. Cancer Surveys, 1:429-449 (1982), which are hereby
incorporated by reference. Procedures for preparing antibody-ricin
conjugates are described in U.S. Pat. No. 4,414,148 and by Osawa, T., et
al. Cancer Surveys, 1:373-388 (1982) and the references cited therein,
which are hereby incorporated by reference. Coupling procedures as also
described in EP 86309516.2, which is hereby incorporated by reference.
Alternatively, the antibody, binding portion thereof, or probe can be
coupled to high energy radiation, e.g., a radioisotope such as 1311,
which, when localized at the tumor site, results in a killing of several
cell diameters. See, e.g., S. E. Order, "Analysis, Results, and Future
Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer
Therapy", Monoclonal Antibodies for Cancer Detection and Therapy, R. W.
Baldwin et al. (eds.), pp 303-316 (Academic Press 1985), which is hereby
incorporated by reference. Radiotherapy is expected to be particularly
effective, because prostate cancer is a relatively radiosensitive tumor.
The antibody or binding portion thereof or probe of the present invention
can be used and sold together with equipment, as a kit, to detect the
particular label.
The therapeutic use of the antibodies, binding portions thereof, or probes
of the present invention can be used in conjunction with other therapeutic
treatment modalities. Such other treatments include surgery, radiation,
cryosurgery, thermotherapy, hormone treatment, chemotherapy, vaccines, and
other immunotherapies.
Also encompassed by the present invention is a method of killing or
ablating which involves using the antibodies, binding portions thereof, or
probes for prophylaxis. For example, these materials can be used to
prevent or delay development or progression of prostate cancer.
Use of the prostate cancer therapy of the present invention has a number of
benefits. Since the antibodies or binding portions thereof or probes
according to the present invention only target prostate epithelial cells,
other tissue is spared. As a result, treatment with such antibodies or
binding portions thereof or probes is safer, particularly for elderly
patients. Treatment according to the present invention is expected to be
particularly effective, because it directs high levels of antibodies or
binding portions thereof or probes to the bone marrow and lymph nodes
where prostate cancer metastases predominate. Moreover, tumor sites for
prostate cancer tend to be small in size and, therefore, easily destroyed
by cytotoxic agents. Treatment in accordance with the present invention
can be effectively monitored with clinical parameters such as serum
prostate specific antigen and/or pathological features of a patient's
cancer, including stage, Gleason score, extracapsular, seminal, vesicle or
perineural invasion, positive margins, involved lymph nodes, etc.
EXAMPLES
Example 1
Human Tissues.
Fresh specimens of benign and malignant tissues were provided by the Tumor
Procurement Service of the Department of Pathology at the Memorial
Sloan-Kettering Cancer Center.
A soluble tissue preparation ("SPTP") was prepared by initial mechanical
mincing of fresh benign and malignant prostates. The tissue was
homogenized for 1 min in a blade homogenizer in phosphate buffered saline
("PBS"), pH 7.2, containing 0.2 mM phenylmethylsulfonyl fluoride (Sigma
Chemical, St. Louis, Mo.) and 20 u/ml aprotinin (Calbiochem, San Diego,
Calif.). The homogenate was centrifuged at 100,000 g for 60 min at
4.degree. C., and the supernatant was decanted and saved.
Example 2
Tissue Culture.
Cultured cell lines of human cancers were from the laboratory of Human
Tumor Immunology, Memorial Sloan-Kettering Cancer Center. The prostate
cancer cell lines PC-3 (Mickey, D. D., et al., "Characterization Of A
Human Prostate Adenocarcinoma Cell Line (DU145) As A Monolayer Culture And
As A Solid Tumor In Athymic Mice," Prog. Clin. Biol. Res., 37:67-84
(1980), which is hereby incorporated by reference), DU-145 (Mickey, D. D.,
et al., "Characterization Of A Human Prostate Adenocarcinoma Cell Line
(DU145) As A Monolayer Culture And As A Solid Tumor In Athymic Mice,"
Prog. Clin. Biol. Res., 37:67-84 (1980), which is hereby incorporated by
reference), and LNCaP (Horoszewicz, J. S., et al., "LNCaP Model Of Human
Prostatic Carcinoma," Cancer Res., 43:1809-1818 (1983), which is hereby
incorporated by reference) were obtained from the American Type Culture
Collection (Rockville, Md.). Hybridomas were initially cloned in RPMI-1640
medium supplemented with 10% FCS, 0.1 mM nonessential amino acids, 2 mM
L-glutamine, 100 units/ml of penicillin, 100 ug/ml of streptomycin and HAT
medium (GIBCO, Grand Island, N.Y.). Subclones were cultured in the same
medium without aminopterin.
Example 3
Preparation of Mouse Monoclonal Antibodies
A BALB/c mouse was immunized subcutaneously with mechanically minced
tissues from fresh benign hyperplastic and malignant prostate tissues
three times at 1 week intervals. One week later, a final intraperitoneal
immunization was administered. Three days later spleen cells were fused
with SP-2 mouse myeloma cells utilizing standard techniques. Ueda, R., et
al., "Cell Surface Antigens Of Human Renal Cancer Defined By Mouse
Monoclonal Antibodies: Identification Of Tissue-Specific Kidney
Glycoproteins," Proc. Natl. Acad. Sci. USA, 78:5122-5126 (1981), which is
hereby incorporated by reference. Supernatants of the resulting clones
were screened by immunohistochemistry. Clones which were reactive with
benign prostate tissues, but not with normal lymph node, were selected and
subcloned 3 times by limiting dilution. The immunoglobulin class of
cultured supernatant from each clone was determined by immunodiffusion
using specified rabbit antisera (Calbiochem, San Diego, Calif.). mAbs were
purified using the MAPS-II kit (Bio-Rad, Richmond, Calif.).
Example 4
Biotinylation of mAbs
Purified mAbs were dialyzed in 0.1M NaCo.sub.3 for 2 hours. One ml of mAb
at 1 mg/ml was mixed with 0.1 ml of biotinamidocaproate
N-hydroxysuccinamide ester (Sigma) 1 mg/ml in dimethylsulfoxide and
stirred for 4 hours at room temperature. Unbound biotin was removed by
dialysis against PBS.
Example 5
Immunohistochemical Staining.
For the initial screening of hybridomas, cryostat sections of prostate
tissues were placed inside rings of Falcon 3034 plate covers
(Becton-Dickenson, Lincoln Park, N.J.) previously coated with 0.45%
gelatin solution. Marusich, M. F., "A Rapid Method For Processing Very
Large Numbers Of Tissue Sections For Immunohistochemical Hybridoma
Screening," J. Immunol. Methods, 111:143-145 (1988), which is hereby
incorporated by reference. Plates were stored at -80.degree. C. Cryostat
sections were fixed with 2% paraformaldehyde in PBS for 10 min at room
temperature and, after washing with PBS, endogenous peroxidase activity
was blocked by treatment with 0.3% hydrogen peroxide in PBS for 10 min at
room temperature. After sections were incubated with 2% BSA in PBS for 20
min, mAbs were added for 60 min at room temperature. Slides were
extensively washed with PBS and incubated with peroxidase-conjugated
rabbit anti-mouse Ig (DAKO Corp., Santa Barbara, Calif.) diluted 1:100 in
10% normal human serum in PBS for 60 min at room temperature. After a
diaminobenzidine reaction, sections were counterstained with hematoxylin.
To confirm cell surface expression of the detected antigens, fresh prostate
tissue was mechanically dispersed into a single cell suspension by
scraping the tissue sample and passing it through a 50 micron sieve. The
cell suspension was washed, incubated with mAb for 1 hour at room
temperature and then a rabbit anti-mouse Ig-fluorescein (DAKO Corp., Santa
Barbara, Calif.). Slides were read with a fluorescent microscope. Negative
control consisted of an isotype-matched irrelevant mAb, while an
anti-class I MHC mAb served as a positive control.
Example 6
Serological Analysis
The anti-mouse immunoglobulin mixed hemadsorption assay was performed as
previously described. Ueda, R., et al., "Cell Surface Antigens Of Human
Renal Cancer Defined By Mouse Monoclonal Antibodies: Identification Of
Tissue-Specific Kidney Glycoproteins," Proc. Natl. Acad. Sci. USA,
78:5122-5126 (1981), which is hereby incorporated by reference. To prepare
the indicator cells, anti-mouse Ig (DAKO Corp.) was conjugated to type 0
human RBC using 0.01% chromium chloride. Serological assays were performed
on cells previously plated in Terasaki plates (Nunc, Denmark). Antibodies
were incubated with target cells at room temperature for 1 hour. Target
cells were then washed and indicator cells added for 1 hour.
Example 7
mAb reactivity to prostatic acid phosphatase ("PAP")
Monoclonal antibody reactivity to prostatic acid phosphatase was assayed by
direct ELISA. Serial dilutions of purified PAP (Calbiochem, La Jolla,
Calif.) were adsorbed onto Terasaki plates overnight at 37.degree. C. The
plates were washed with PBS 0.5% BSA. PBS 2% BSA was incubated for 60 min
at 37.degree. C. to block non-specific binding. Biotinylated mAb was
incubated for 45 min at room temperature. Rabbit anti-PAP (Sigma, St.
Louis, Mo.) diluted 1/6000 in PBS 2% BSA served as positive control.
Rabbit anti-PAP was followed by biotin-conjugated goat anti-rabbit Ig
(Sigma) 1/5000 in PBS 2% BSA. Avidin-conjugated alkaline phosphatase Sigma
1/500 in PBS 2% BSA for 45 min at room temperature followed biotinylated
antibody. A substrate of alkaline phosphatase (para nitrophenylphosphate)
was incubated at 37.degree. C., and reactivity was read at OD.sub.405 nm
on an Artek ELISA reader adapted for Terasaki plates. Negative controls
omitted PAP antigen and/or Rabbit anti-PAP.
Example 8--mAb reactivity to prostate specific antigen ("PSA").
Monoclonal antibody reactivity to prostate specific antigen was assayed by
a double antibody sandwich ELISA. Terasaki plates were coated with rabbit
anti-PSA (Accurate Chemical and Scientific Corp, N.Y.) diluted 1/1000 in
carbonate coating buffer overnight at 37.degree. C. PBS 2% BSA was used to
block non-specific binding. The soluble prostate tissue preparation
("SPTP") provided a source of PSA. SPTP was serially diluted in PBS 2% BSA
and incubated at RT for 45 min. Biotinylated mAbs were added for 45 min.
Avidin-conjugated alkaline phosphatase and substrate were used as
described above for the direct ELISA. Negative controls omitted the Rabbit
anti-PSA capture antiserum or the PSA (SPTP).
Example 9
Immunoprecipatation
SPTP was applied to a Concanavalin A column and eluted by 0.2M a-methyl
D-mannoside. Fractions containing PSA were determined by a sandwich ELISA
using Rabbit anti-PSA and biotin-conjugated Prost 410. Pooled PSA
fractions were labelled with I-125 by the chloramide-T method. Unbound
I-125 was removed with a PD10 column (BIO-RAD, Richmond, Calif.). Labelled
antigen was precleared by normal mouse or rabbit sera once and
precipitated with mAbs or polyclonal antibodies and protein A sepharose
(Boehringer Manheim Biochem.) For sequential immunoprecipitations,
labelled antigens were precleared with normal serum, precleared 3 times by
first antibodies. Resulting supernatants were precipitated with second
antibodies and protein A sepharose. Each precipitate was applied to 9%
SDS-PAGE by the method of Laemmli. Laemmli, U.K., "Cleavage Of Structural
Proteins During The Assembly Of The Head Of Bacteriophage T4," Nature
(London), 227:680-685 (1970), which is hereby incorporated by reference.
Approximately 800 clones resulted from this fusion, of which six clones
were initially selected based on immunohistochemical reactivity with
prostate epithelium and the absence of reactivity with lymph node tissue.
After subcloning, supernatants from the 6 hybridomas were assayed on a
panel of cell lines using a mixed hemadsorption assay (Table 3).
TABLE 3
__________________________________________________________________________
Reactivity of mAbs with human cell lines by
rabbit anti-mouse Ig rosetting assay
Prost 16
Prost 30
Prost 130
Prost 185
Prost 284
Prost 410
Cell lines (.gamma..sup.1)
(.gamma..sup.1)
(.gamma..sup.1)
(.gamma..sup.1)
(.mu.)
(.gamma..sup.1)
__________________________________________________________________________
Renal
SK-RC-18, 39, 4, 53, 42,
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8, 26, 31, 45, 48
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1, 59, 21, 2, 44,
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47, 62, 28, 9, 17
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Bladder
VmCUB-1, -2, 647V, RT4
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253J, 5637, 639V, T234
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PC-3, Du145, LNCaP
Melanoma
SK-MEL-23, 28, 31, 37
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Astrocytoma
SK-MG-1, 4, 5, 7
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Colon
Sw1116, Sw480, HCT15,
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HT29
LS174T, SK-CO-11,
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.smallcircle..smallcircle..smallcircle.
.smallcircle..smallcircle..smallcircle.
.smallcircle..smallcircle..smallcircle.
.smallcircle..smallcircle..smallcircl
e.
SK-CO-17
Lung .smallcircle.
.smallcircle.
.smallcircle.
.smallcircle.
.smallcircle.
.smallcircle.
HCIH69
Hematopoietic
SK-Ly-18, -16, DAUDI
.smallcircle..smallcircle..smallcircle.
.smallcircle..smallcircle..smallcircle.
.smallcircle..smallcircle..smallcircle.
.smallcircle..smallcircle..smallcircle.
.smallcircle..smallcircle..smallcircle.
.smallcircle..smallcircle..smallcircl
e.
BALL-1, HL-60, SK-DHL-2
.smallcircle..smallcircle..smallcircle.
.smallcircle..smallcircle..smallcircle.
.smallcircle..smallcircle..smallcircle.
.smallcircle..smallcircle..smallcircle.
.smallcircle..smallcircle..smallcircle.
.smallcircle..smallcircle..smallcircl
e.
U937, RAMOS, RAJI
.smallcircle..smallcircle..smallcircle.
.smallcircle..smallcircle..smallcircle.
.smallcircle..smallcircle..smallcircle.
.smallcircle..smallcircle..smallcircle.
.smallcircle..smallcircle..smallcircle.
.smallcircle..smallcircle..smallcircl
e.
HSB2 .smallcircle.
.smallcircle.
.smallcircle.
.smallcircle.
.smallcircle.
.smallcircle.
Pancreas .circle-solid.
.smallcircle.
.smallcircle.
.smallcircle.
.circle-solid.
.smallcircle.
ASPC-1
__________________________________________________________________________
Prost 16 and Prost 284 showed virtually identical reactivities; as Prost
284 was an IgM, it was put aside in favor of Prost 16, an IgG.sub.1. Prost
410 reacted only with LNCaP, and Prost 30, Prost 130, and Prost 185 failed
to react with any cell lines including the prostate cancer cell lines
PC-3, DU 145, and LNCaP. After purification of the 5 selected mAbs using
protein A columns, reactivities of these mAbs on normal human tissues were
examined immunohistochemically (Table 4).
TABLE 4
______________________________________
Reactivity of mAbs with human normal tissues
by indirect immunoperosidase staining
Prost 16
Prost 30
Prost 130
Prost 185
Prost 410
Tissues (.gamma..sup.1)
(.gamma..sup.1)
(.gamma..sup.2a)
(.gamma..sup.1)
(.gamma..sup.1)
______________________________________
Prostate .circle-solid.
.circle-solid.
.circle-solid.
.circle-solid.
.circle-solid.
Kidney
Glomerulus
.largecircle.
.largecircle.
.largecircle.
.largecircle.
.largecircle.
Tubule .circle-solid.
.box-solid.
.box-solid.
.box-solid.
.largecircle.
Ureter .circle-solid.
.largecircle.
.largecircle.
.largecircle.
.largecircle.
Bladder .circle-solid.
.largecircle.
.largecircle.
.largecircle.
.largecircle.
Testis .circle-solid.
.largecircle.
.largecircle.
.largecircle.
.largecircle.
Uterus
Cervix .circle-solid.
.largecircle.
.largecircle.
.largecircle.
.largecircle.
Endometrium
.circle-solid.
.largecircle.
.largecircle.
.largecircle.
.largecircle.
Fallopian tube
.circle-solid.
.largecircle.
.largecircle.
.largecircle.
.largecircle.
Placenta .circle-solid.
.largecircle.
.circle-solid.
.circle-solid.
.largecircle.
Umbilical cord
.largecircle.
.largecircle.
.largecircle.
.largecircle.
.largecircle.
Cerebrum .largecircle.
.largecircle.
.largecircle.
.largecircle.
.largecircle.
Cerebellum
.largecircle.
.largecircle.
.largecircle.
.largecircle.
.largecircle.
Thymus .circle-solid.
.largecircle.
.circle-solid.
.circle-solid.
.largecircle.
Parotid gland
.circle-solid.
.largecircle.
.circle-solid.
.circle-solid.
.largecircle.
Breast .circle-solid.
.largecircle.
.largecircle.
.largecircle.
.largecircle.
Lung
Alveola .largecircle.
.largecircle.
.largecircle.
.largecircle.
.largecircle.
Bronchiole
.circle-solid.
.largecircle.
.largecircle.
.largecircle.
.largecircle.
Stomach .circle-solid.
.largecircle.
.largecircle.
.largecircle.
.largecircle.
Colon .circle-solid.
.largecircle.
.circle-solid.
.circle-solid.
.largecircle.
Pancreas .circle-solid.
.largecircle.
.largecircle.
.largecircle.
.largecircle.
Liver .largecircle.
.largecircle.
.largecircle.
.largecircle.
.largecircle.
Adrenal gland
.largecircle.
.largecircle.
.largecircle.
.largecircle.
.largecircle.
Lymph node
.largecircle.
.largecircle.
.largecircle.
.largecircle.
.largecircle.
Skin .circle-solid.
.largecircle.
.largecircle.
.largecircle.
.largecircle.
Foreskin .circle-solid.
.largecircle.
.circle-solid.
.largecircle.
.largecircle.
______________________________________
.circle-solid. -- positive; .box-solid. -- weak, heterogeneous;
.largecircle. -- negative
Prost 16 showed broad reactivity and was not further characterized. Prost
130 and Prost 185 showed relatively restricted and almost identical
reactivities. Prost 30 and Prost 410 showed highly restricted
reactivities. None of these 5 mAbs demonstrated immunohistochemical
reactivity with normal rat prostate nor the Dunning R-3327 rat prostate
cancer cell line.
mAb Prost 30: Purified Prost 30 (40 ug/ml) did not react, by MHA, with any
of an expanded panel of 74 human cell lines. By indirect immunoperoxidase
assays, Prost 30 also failed to react with any of 29 cell lines (including
LNCaP, PC-3, and DU 145) after 20 paraformaldehyde fixation.
Immunohistochemical study of frozen tissue sections revealed all 35 benign
and 30 malignant prostates were Prost 30-positive (FIG. 1). Prost 30
reacted with the prostatic epithelial cells and luminal secretions. No
other tissues tested were reactive except for weak and heterogeneous
reactivity with some tubules in 7 of 19 normal kidney specimens and 1 of 7
lung cancers (Tables 5 and 6).
TABLE 5
______________________________________
Immunohistochemical reactivity of mAbs
with human normal tissues
Reactivity
Prost 30 Prost 130
Prost 185
______________________________________
Prostate .sup. 35/35.sup.a
35/35 35/35
Kidney 7/19 3/10 3/10
Lung 0/6 0/4 0/4
Liver 0/6 0/6 0/5
Spleen 0/4 0/3 0/3
Thymus 0/1 1/1 1/1
Ureter 0/9 0/5 0/5
Bladder 0/10 0/8 0/8
Testis 0/3 0/3 0/3
Breast 0/7 2/5 2/5
Esophagus 0/1 1/1 1/1
Stomach 0/3 2/2 1/2
Small intestine
0/2 1/2 1/2
Colon 0/4 3/4 2/4
Pancreas 0/1 0/1 0/1
Uterus 0/4 0/2 0/2
Thyroid 0/2 1/1 1/1
Adrenal 0/3 0/2 0/2
Parotid 0/2 2/2 2/2
Submandibular gland
0/1 1/1 1/1
Skin 0/2 0/2 0/2
Cerebrum 0/1 0/1 0/1
Cerebellum 0/1 0/1 0/1
______________________________________
.sup.a Number of specimens with positive staining/number of specimens
tested.
Based on relative endpoint titrations with all 3 mAbs, immunoreactivity o
prostate tissue was 200-500 fold that on other positive tissues.
TABLE 6
______________________________________
Immunohistochemical reactivity of mAbs
with human cancers
Reactivity
Cancer Prost 30 Prost 130
Prost 185
______________________________________
Prostate .sup. 30/30.sup.a
30/30 30/30
Renal 0/17 0/7 0/7
Bladder 0/21 0/8 0/8
Lung 1/7 1/4 1/4
Breast 0/6 2/6 2/6
Colon 0/5 3/6 2/6
Ovary 0/6 2/6 0/6
Testis 0/2 n.t. n.t.
______________________________________
.sup.a Number of specimens with positive staining/number of specimens
tested.
Based on relative endpoint titrations with all 3 mAbs, immunoreactivity o
prostate tissue was 200-500 fold that on other positive tissues.
n.t. = not tested
mAb Prost 30 failed to react with paraffin sections. Immunofluorescence
assay of fresh, viable prostate cells demonstrated cell surface
fluorescence. The antigen recognized by Prost 30 was heat sensitive and
resistant to treatment with 20 mM sodium periodate. Prost 30 did not react
with either PSA or PAP by ELISA.
mAbs Prost 130 and Prost 185: As previously noted, mAbs Prost 130 and Prost
185 had virtually identical reactivity versus cell line targets (i.e.,
non-reactive; see Table 3) and tissue sections (Tables 5 and 6; see FIG.
2). While qualitatively not as tissue-specific as Prost 30, Prost 130 and
185 were quantitatively quite specific. That is, the IHC endpoint titers
of Prost 130 and 185 were 200-500 fold higher on prostate tissue than on
other IHC-reactive tissues. Like Prost 30, mAbs Prost 130 and 185 also
failed to react with paraffin sections. Immunofluorescence assay of fresh,
viable prostate cells demonstrated cell surface fluorescence. Both mAbs
were reactive against SPTP by direct ELISA. Using a double antibody
sandwich ELISA, antigen captured by Prost 130 was reactive with either
Prost 130-biotin or Prost 185-biotin, but not with Prost 410-biotin, as a
second antibody (FIG. 3A). Conversely, antigen captured using Prost 185 as
a first antibody was reactive with Prost 130-biotin but non-reactive with
either Prost 185-biotin or Prost 410-biotin as a second antibody (FIG.
3B). These results suggest that Prost 130 and Prost 185 recognize the same
molecule, that this molecule has at least two Prost 130-reactive epitopes
but only a single Prost 185-reactive epitope, and the antigen is not PSA.
To confirm the Prost 130 and Prost 185 epitopes were different, a double
antibody sandwich competitive ELISA was performed (Fig.. 4). Antigen from
SPTP was captured by Prost 130. Unconjugated mAbs Prost 130, Prost 185,
and Prost 410 were added to compete for binding by Prost 130-biotin (FIG.
4A). Only Prost 130, but neither Prost 185 or Prost 410, could inhibit
Prost 130-biotin binding. Similarly, only Prost 185, but neither Prost 130
nor Prost 410, could inhibit Prost 185-biotin (FIG. 4B).
mAb Prost 410: Using the rabbit anti-mouse Ig MHA and an ELISA assay,
purified Prost 410 at 40 .mu.g/ml was reactive only with the LNCaP line of
83 human cell lines tested. By immunohistochemistry, it reacted with all
normal, hyperplastic and neoplastic prostatic tissue sections tested (FIG.
1, Table 5). A sandwich ELISA assay demonstrated reactivity of Prost 410
to prostate specific antigen ("PSA"). The PSA reactivity of Prost 410 was
confirmed by immunoprecipitation.
Previous efforts to develop mAbs to prostate-related molecules have been
directed either toward previously characterized molecules of prostatic
origin such as PAP or PSA or toward defining antigens which distinguish
prostate cancer from normal or hyperplastic prostatic epithelium (i.e.,
BPH). In this study, a different approach was taken. A need for improved
imaging of regional nodes was identified as a clinically valuable goal as
this area represented such a common site of metastatic spread and yet one
which has proven difficult to assess without a surgical staging procedure.
The objective was to develop mAbs for use in clinical imaging of prostate
cancer within the regional (pelvic) lymph nodes. A number of assumptions
were made. First, the mAb need not specifically distinguish prostate
cancer from BPH or normal prostate, because the presence of prostate
antigen-expressing cells within a lymph node is, by definition, metastatic
prostate cancer. It was felt that this broadening of the specificity
requirement would substantially increase our likelihood of success.
Secondly, administration of the mAb to patients via a selective, rather
than a systemic, route (e.g., intra or periprostatic injection or via
subcutaneous injection of the lower extremity was also anticipated). Both
animal (Weinstein, J. N., et al., "Monoclonal Antibodies In The
Lymphatics: Toward The Diagnosis And Therapy Of Tumor Metastases,"
Science, 218:1334-1337 (1982); Weinstein, J. N., et al., "Monoclonal
Antibodies In The Lymphatics: Selective Delivery To Lymph Node Metastases
Of A Solid Tumor," Science, 222:423-426 (1983); Parker, R. J., et al.,
"Targeting Of Murine Radiolabeled Monoclonal Antibodies In The
Lymphatics," Cancer Res., 47:2073-2076 (1987), which are hereby
incorporated by reference) and human (Keenan, A. M., et al.,
"Immunolymphoscintigraphy In Patients With Lymphoma After Subcutaneous
Injection Of Indium-111-Labeled T101 Monoclonal Antibody," J. Nucl. Med.,
28:42-46 (1987); Keenan, A. M., et al., "Immunolymphoscintigraphy And The
Dose-Dependence Of Indium 111-Labeled T101 Monoclonal Antibody In Patients
With Cutaneous T-Cell Lymphoma," Cancer Res., 47:6093-6099 (1987), which
are hereby incorporated by reference). Studies have shown significant
potential advantage by juxtaposing such anatomic selectivity to the
inherent antigenic specificity of the mAb. The anticipated regional
administration, therefore, allowed further liberalization of the mAbs'
specificity requirement. This setting made it reasonable to screen and
select clones simply on the basis of prostate reactivity in the absence of
nodal reactivity.
Among the mAbs produced in this study, Prost 30, 130, and 185 appear
different from previously defined prostate-related mAbs. For instance,
mAbs PD41 (Beckett, M. L., et al., "Monoclonal Antibody PD41 Recognizes An
Antigen Restricted To Prostate Adenocarcinomas," Cancer Res., 51:1326-1333
(1987), which is hereby incorporated by reference), P25.48 and P25.91
(Bazinet, M., et al., "Immunohistochemical Characterization Of Two
Monoclonal Antibodies, P25.48 And P25.91, Which Define A New
Prostate-Specific Antigen," Cancer Res., 48:6938-6942 (1988), which is
hereby incorporated by reference), and P6.2 (Wright, G. L., Jr., et al.,
"Immunohistochemical Localization Of Prostate Carcinoma-Associated
Antigens," Cancer Res., 43:5509-5516 (1983), which is hereby incorporated
by reference) define antigens restricted to a subset of prostate cancers
but not expressed by either normal or hyperplastic prostatic epithelial
cells. Among the mAbs which define antigens shared by normal,
hyperplastic, and neoplastic prostatic cells, clone 35 (Frankel, A. E., et
al., "Monoclonal Antibodies To Human Prostate Antigen," Cancer Res.,
42:3714-3718 (1982), which is hereby incorporated by reference), differs
from those mAbs reported here by virtue of clone 35's reactivity with
breast epithelium and bladder cancer cell line T-24. When clone 35 is
assayed by a membrane radioimmunoassay ("RIA"), it is more reactive with
normal kidney than prostate tissue. Clone 24 (Frankel, A. E., et al.,
"Monoclonal Antibodies To Human Prostate Antigen," Cancer Res.,
42:3714-3718 (1982), which is hereby incorporated by reference), is
reactive with the PC-3 cell line and, in a membrane RIA, demonstrated high
reactivity to BPH but only background reactivity with prostate cancer. mAb
.alpha.Pro3 (Ware, J. L., et al., "Production Of Monoclonal Antibody
.alpha.Pro3 Recognizing A Human Prostatic Carcinoma Antigen," Cancer Res.,
42:1215-1222 (1982), which is hereby incorporated by reference), bound
PC-3 cells, and, although Immunohistochemistry was not performed, an
absorption assay utilizing tissue extracts appears to indicate greater
antigen expression in a wide range of non-prostatic tissues than in BPH.
The epitopes detected by mAbs F77 (Carroll, A. M., et al., "Monoclonal
Antibodies To Tissue-Specific Cell Surface Antigens," Clin. Immunol. And
Immunopathol., 33:268-281 (1984), which is hereby incorporated by
reference), KR-P8 (Raynor, R. H., et al., "Characterization of a
Monoclonal Antibody, KR-P8, That Detects A New Prostate-Specific Marker,"
J. Natl. Cancer Inst., 73:617-625 (1984); Raynor, R. H., et al.,
"Biochemical Nature Of The Prostate-Associated Antigen Identified By The
Monoclonal Antibody," KR-P8, Prostate, 9:21-31 (1986), which are hereby
incorporated by reference), TURP-27 and TURP-73 (Starling, J. J., et al.,
"Human Prostate Tissue Antigens Defined By Murine Monoclonal Antibodies,"
Cancer Res., 46:367-374 (1986), which is hereby incorporated by reference)
are detectable on formalin fixed/paraffin embedded tissue sections unlike
either Prost 30, 130, or 185. The TURP-73 antigen is also detectable on
several prostate cancer cell lines. One previously reported mAb, 7E11-C5
(Horoszewicz, J. S., et al., "Monoclonal Antibodies To A New Antigenic
Marker In Epithelial Prostatic Cells And Serum Of Prostatic Cancer
Patients," Anticancer Res., 7:927-936, (1987), which is hereby
incorporated by reference), has some characteristics similar to Prost 30,
130, and 185. These similarities include lack of reactivity with cell
lines, weak immunohistochemical reactivity with some renal tubules, and
reactivity with all normal, BPH, and neoplastic prostates tested. There
are, however, features which differ: 7Ell-C5 reacts with LNCap cells after
fixation, it has immunohistochemical reactivity with skeletal muscle
(Lopes, A. D., et al., "Immunohistochemical And Pharmacokinetic
Characterization Of The Site-Specific Immunoconjugate CYT-356 Derived From
Antiprostate Monoclonal Antibody 7E11-C5, " Cancer Res., 50:6423-6429,
(1990), which is hereby incorporated by reference) and, at least in the
initial report, the presence of the 7E11-C5 Ag in serum (Horoszewic, J.
S., et al., "Monoclonal Antibodies To A New Antigenic Marker In Epithelial
Prostatic Cells And Serum Of Prostatic Cancer Patients," Anticancer Res.,
7:927-936, (1987), which is hereby incorporated by reference.
Among the previously published mAbs, some have already begun clinical
evaluation for imaging prostate cancer (Vihko, P., et al., "Radioimaging
Of Prostatic Carcinoma With Prostatic Acid Phosphatase--Specific
Antibodies," Biotechnology In Diagnostics, pp. 131-134 (1985); Babaian, R.
J., et al., "Radioimmunological Imaging Of Metastatic Prostatic Cancer
With 111-Indium-Labeled Monoclonal Antibody PAY 276", J. Urol.,
137:439-443 (1987); Leroy, M., et al., "Radioimmunodetection Of Lymph Node
Invasion In Prostatic Cancer. The Use Of Iodine 123 (123-I) -Labeled
Monoclonal AntiProstatic Acid Phosphatase (PAP) 227 A F(ab')2 antibody
Fragments In Vivo," Cancer, 64:1-5 (1989); Meyers, J. F., et al.,
"Development Of Monoclonal Antibody Imaging Of Metastatic Prostatic
Carcinoma," The Prostate, 14:209-220 (1989), which are hereby incorporated
by reference). For example, mAbs to PSA have been used for imaging without
apparent success (Meyers, J. F., et al., "Development Of Monoclonal
Antibody Imaging Of Metastatic Prostatic Carcinoma," The Prostate,
14:209-220, (1989), which is hereby incorporated by reference). Given the
nature of the PSA antigen, this is probably not surprising. While PSA is
very tissue-specific, the antigen is primarily cytoplasmic with little, if
any, cell surface expression (Warhol, J. J., et al., "The Ultrastructural
Localization Of Prostatic Specific Antigen And Prostatic Acid Phosphatase
In Hyperplastic And Neoplastic Human Prostates," J. Urol., 134:607-613
(1985), which is hereby incorporated by reference). Furthermore, PSA is
secreted and can be detected in serum. Systemic administration of antibody
to PSA would be expected to result in immune complex formation, uptake in
the reticuloendothelial system and consequent background imaging.
mAbs to PAP have also been studied for imaging (Vihko, P., et al.,
"Radioimaging Of Prostatic Carcinoma With Prostatic Acid
Phosphatase--Specific Antibodies," Biotechnology In Diagnostics, pp.
131-134, (1985); Leroy, M., et al., "Radioimmunodetection Of Lymph Node
Invasion In Prostatic Cancer. The Use Of Iodine 123 (123-I)-Labeled
Monoclonal Anti-Prostatic Acid Phosphatase (PAP) 227 A F(ab')2 Antibody
Fragments In Vivo," Cancer, 64:1-5, (1989), which are hereby incorporated
by reference). While PAP has features similar to PSA such as being
primarily a cytoplasmic, secreted antigen, trials using a regional, i.e.,
periprostatic injection have claimed initial success (Leroy, M., et al.,
"Radioimmunodetection Of Lymph Node Invasion In Prostatic Cancer. The Use
Of Iodine 123 (123-I) -Labeled Monoclonal Anti-Prostatic Acid Phosphatase
(PAP) 227 A F(ab')2 Antibody Fragments In Vivo," Cancer, 64:1-5 (1989),
which is hereby incorporated by reference). Perhaps, the shortcomings of
such an antigenic target may be overcome by selective/regional
administration.
The antibodies Prost 130 and Prost 185 appear worthy of study via such a
selective site administration. These antibodies target at least 3 epitopes
on the detected antigen, the antigen is strongly expressed at the cell
surface and it does not circulate. The normal tissues which express Prost
130/Prost 185 (thymus, parotid, colon, foreskin, and placenta) should not
present a significant practical problem.
Another mAb currently being evaluated for use in imaging, as well as
therapy, is CYT-356 (Lopes, A. D., et al., "Immunohistochemical And
Pharmacokinetic Characterization Of The Site-Specific Immunoconjugate
CYT-356 Derived From Antiprostate Monoclonal Antibody 7E11-C5, " Cancer
Res., 50:6423-6429 (1990); Wynant, G. E., "Immunoscintigraphy Of Prostatic
Cancer: Preliminary Results With .sup.111 In-Labeled Monoclonal Antibody
7E11-C5.3 (CYT-356), The Prostate, 18:229-241" (1991), which are hereby
incorporated by reference), a subclone of 7E11-C5 (Horoszewicz, J. S., et
al., "Monoclonal Antibodies To A New Antigenic Marker In Epithelial
Prostatic Cells And Serum Of Prostatic Cancer Patients," Anticancer Res.,
7:927-936 (1987); Lopes, A. D., "Immunohistochemical And Pharmacokinetic
Characterization Of The Site-Specific Immunocojugate CYT-356 Derived From
Antiprostate Monoclonal Antibody 7E11-C5," Cancer Res., 50:6423-6429
(1990), which are hereby incorporated by reference). As noted, there are
some similarities between this mAb and Prost 30. Initial imaging results
with CYT-356 appear promising (Wynant, G. E., et al., "Immunoscintigraphy
Of Prostatic Cancer: Preliminary Results With .sup.111 In-Labeled
Monoclonal Antibody 7E11-C5.3 (CYT-356)," The Prostate, 18:229-241 (1991),
which is hereby incorporated by reference).
mAb Prost 30 appears to have some optimal characteristics for localization
to normal and neoplastic prostate either by regional or systemic
administration. Indeed, mAb Prost 30 shares many features with another
antibody--mAb G250--which has already been demonstrated to be successful
in clinical trials of patients with renal cancer. See Oosterwijk, E., et
al., "Antibody Localization In Human Renal Cell Carcinoma: A Phase I Study
Of Monoclonal Antibody G250," J. Of Clin. Oncol., 11:738-750 (1993), which
is hereby incorporated by reference). These common features include
isotype (.gamma..sub.1), a high degree of specificity by
immunohistochemistry, cell surface expression and absence of circulating
antigen. With G250, specific, high level accumulation in both primary and
metastatic renal cancer sites in the absence of normal tissue uptake has
been demonstrated. The immunoscintigraphy study demonstrated high
sensitivity (3 of 12 patients had sites of disease detected on mAb G250
scans which were not diagnosed by conventional studies) and high (100%)
specificity--all mAb detected sites have been histopathologically
confirmed renal cancers. The potential for mAb localization to metastatic
prostate cancer sites may provide utility not only in diagnostic
immunoscintigraphy but also for antibody directed therapy of metastatic
disease. Potential localization to normal or hyperplastic prostate should
not represent a significant problem. Indeed, this might be viewed as an
advantage. If it can be shown that Prost 30 localizes well to the
prostate, the antibody could have clinical potential for treatment of
localized carcinoma of the prostate (alone or in combination with other
therapies), in the treatment of BPH, or even in the prevention of BPH or
prostate cancer.
Example 10
Clinical Data
Fifteen patients with a diagnosis of prostate cancer have received .sup.131
Iodine (10 mCi)-labeled mab Prost 30 intravenously 1 week prior to either
surgery (i.e., radical prostatectomy) or biopsy of a suspicious lesion. In
the week between Prost 30 injection and surgery/biopsy, patients underwent
whole body radionuclide scanning on 2-3 occasions and one SPECT scan.
Successive patients, in cohorts of 3, received escalating doses of Prost
30 (all with 10 mCi .sup.131 Iodine): 1.0 mg (3 patients), 2.0 mg (3
patients), 5.0 mg (3 patients), 10.0 mg (3 patients), and 20.0 mg (3
patients).
Of the 15 patients, 14 had a prostate gland in situ. In all of these cases,
the prostate gland could be visualized on the whole-body and spect images.
The 1 patient without a prostate in situ showed no Prost 30 localization
to the prostatic bed, demonstrating specificity and absence of
false-positives. Two patients had demonstrable metastatic disease by
conventional CT scans. In both cases, the monoclonal antibody images
visualized these sites (one patient: lymph nodes plus liver; second
patient: lymph nodes). In 3 cases, the resected prostate specimen was
scanned/imaged alongside tubes of blood drawn at the time of resection.
These images (see FIG. 5) demonstrate substantial specific and selective
accumulation and concentration of the labeled Prost 30 in the prostate
(target site of disease) relative to the blood or other normal tissues.
This indicates that wherever prostate cells may be in the body (e.g.,
lymph node, bone marrow, etc.), Prost 30 will bind to those cells.
Two patients entered in the above study had progressive hormone-refractory
disease with rising prostate specific antigens ("PSA") prior to entry.
Subsequent to Prost 30 administration, their PSAs reversed course and
dropped substantially (by approx 75%). The PSAs did not return to
pre-treatment baseline levels for 9-10 months. Three patients who were not
previously treated with hormonal therapy were given hormonal therapy
shortly after Prost 30. Their PSAs have fallen to and remained at
undetectable levels. Five patients, including 4 at high risk of relapse
(i.e., with high pre-treatment PSA and adverse pathological features) had
Prost 30 plus surgery. None of these patients have yet relapsed. One
patient received Prost 30 followed by radiation therapy. Although at high
risk of failure given his pre-treatment PSA, imaging studies and biopsy
results, he too remains a complete responder with undetectable serum PSA
levels and no evidence of disease. The above results indicate that the
Prost 30 antibody itself has a therapeutic effect. Only 1 mg of the
administered dose was actually labeled with .sup.131 I, while the balance
of the administered dose (0 to 19 mg) did not contain iodine. The .sup.131
Iodine label attached to the Prost 30 monoclonal antibody is simply a
tracer dose in a quantity insufficient to explain the therapeutic effect.
Two of seven patients did not develop any evidence of human anti-mouse
antibody (HAMA) formation, as defined by a very sensitive assay, after
Prost 30 treatment. The 2 hormone-refractory patients did not develop
HAMA.
A phase I/II therapy trial with unconjugated Prost 30 has begun. The first
2 patients entered are also showing PSA declines, indicating therapeutic
benefit.
Example 11
C37 and C219 Monoclonal Antibodies
BALB/c mice were immunized once with a cell suspension of the LNCaP human
prostate cancer cell line. Approximately four days later, the mice were
sacrificed and spleens harvested for preparation of hybridomas. This
immunization design is optimized for production of IgM antibodies which
are the strongest at complement fixation (i.e., they are the best at
mediating complement lysis of target cells). IgMs are often avoided in
monoclonal work because they are very large molecules (5-10.times.larger
than IgGs), and there are concerns about them being able to penetrate into
tumor deposits. This could be an advantage, because the predominant site
of metastatic disease is bone marrow and lymph nodes which should be
readily accessible to IgMs. Conversely, normal tissues will be exposed to
lower levels of these IgMs decreasing the chances of cross-reaction.
Candidate antibodies were screened and selected using a complement fixation
assay with the immunizing cell line (LNCaP) as a target. That is, the
hybridoma supernatants were incubated with the target cells in the
presence of human serum (i.e. the source of complement) and the hybridomas
whose supernatants lysed/killed the target cells were selected. Any
antibodies which also lysed non-prostate cells were not selected. As a
result, two clones, designated C37 and C219, which are very potent and
specific at lysing LNCaP, were identified. Furthermore, when they were
combined, these antibodies did not function in an additive manner, but in
a synergistic one.
An approach that uses cytotoxic mechanisms, such as complement, has
inherent advantages over methods which use conjugated agents. It avoids
the necessity of linking an agent to the antibody, such conjugation is a
developing science unto itself. It also eliminates all of the issues
related to how those agents kill the cells. For example, the concept of
using radioisotopes not only is complicated due to the linkage issues, but
so is the science of radio emitters. Does one use alpha, beta, or gamma
emitters? Does one need internalized or non-internalized antigens? Using
these endogenous cytotoxic mechanisms such as complement-mediated
cytoxicity also eliminates the side effects of conjugated agents. It also
makes handling and preparation of the therapeutic dramatically simpler.
Furthermore, the complement system is itself a self-amplifying one. That
is, as each successive enzyme in the cascade is activated, it, in turn,
activates many more molecules and becomes an amplified process.
Part of the effect of triggering the complement system is that it also
recruits leukocytes, including immune cells, into the area by release of
chemotactic factors. As a result, the complement system generates quite a
substantial and amplified immune response, both cellular and humoral, with
the use of antibody alone as the triggering mechanism.
Although the invention has been described in detail for the purpose of
illustration, it is understood that such detail is solely for that purpose
and variations can be made by those skilled in the art without departing
from the spirit and scope of the invention which is defined by the
following claims.
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